1. NoRC, a novel nucleolar chromatin remodeling complex

Potentially active rRNA genes exhibit an ‚open’ chromatin structure and are characterized by DNA hypomethylation, and euchromatic histone marks. Epigenetically silenced rRNA genes are demarcated by CpG hypermethylation (CH3), heterochromatic histone marks (Me) and association with heterochromatin protein 1 (HP1).
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rRNA genes (rDNA) exist in two distinct types of chromatin, an ‘open’ one that is permissive to transcription and a ‘closed’ one that is transcriptionally refractive. The mechanisms that maintain the ratio of active vs. silent rDNA repeats are poorly understood. Our research aims at understanding the principles that establish and propagate the active and silent state of rRNA genes.

NoRC coordinates rDNA silencing through several epigenetic layers.
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We have identified and functionally characterized NoRC (Nucleolar Remodeling Complex), a novel member of SNF2h/ISWI-containing chromatin remodeling complexes. NoRC recruits DNA methyltransferase and histone deacetylases and methyltransferases to the rDNA promoter, thereby triggering heterochromatin formation. NoRC-mediated changes in chromatin structure lead to repression of rDNA transcription and to resetting replication timing from early to late. It seems that NoRC serves at least two functions: first, as a remodeling complex that alters the nucleosome position at the rDNA promoter; and second, as a scaffold that coordinates the activities of macromolecular complexes that modify histones, methylate DNA and establish a ’closed’ heterochromatic chromatin state.

2. Long noncoding RNAs guide chromatin regulators to specific genomic loci

Recruitment of chromatin modifiers by DNA:RNA triplex structures
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Nuclear long noncoding RNAs (lncRNAs) have been implicated in most, if not all, chromatin-mediated processes, but few of these lncRNAs have been ascribed functions and very little is known about their mode of action and regulation. We found a lncRNA that interacts with and targets the chromatin remodeling complex NoRC to the rDNA promoter. Mechanistically, pRNA forms a transient DNA:RNA triplex with a regulatory site, which is specifically recognized by the de novo DNA methyltransferase DNMT3b (Schmitz et al. 2010). This finding reveals a compelling new mechanism of RNA-dependent epigenetic regulation implying that DNA:RNA triplexes may serve as anchor points or be used as landmarks to guide chromatin regulators to specific genomic loci to regulate expression of target genes. Our current research focuses on identifying such novel target genes.

 

3. Regulation of rDNA transcription by antisense RNA

© Repression of rDNA transcription by antisense RNA mediated recruitment of the histone methyltransferase Suv4-20h2 and histone H4K20 trimethylation during cell quiescense.

Global transcriptome analysis has shown that up to 70% of the sense transcripts have antisense partners and that antisense RNA can alter the expression of the respective sense gene. Strand-specific PCR-walking across rDNA revealed low levels of antisense transcripts, termed PAPAS ('promoter and pre-rRNA antisense') that cover sequences of pre-rRNA and the rDNA promoter. Importantly, the level of PAPAS shows reverse correlation to pre-rRNA, i.e., PAPAS being upregulated in growth-arrested cells and downregulated in cancer cells. PAPAS guide the H4K20 histone methyltransferase Suv4-20h2 to rDNA leading to compaction of chromatin, which in turn impairs transcription and enforces signal-dependent transcriptional shutdown in response to growth arrest. 

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